Long non-coding RNA GRASLND enhances chondrogenesis via suppression of the interferon type II signaling pathway.

The roles of long noncoding RNAs (lncRNAs) in musculoskeletal development, disease, and regeneration remain poorly understood. Here, we identified the novel lncRNA GRASLND (originally named RNF144A-AS1) as a regulator of mesenchymal stem cell (MSC) chondrogenesis. GRASLND, a primate-specific lncRNA, is upregulated during MSC chondrogenesis and appears to act directly downstream of SOX9, but not TGF-ß3. We showed that the silencing of GRASLND resulted in lower accumulation of cartilage-like extracellular matrix in a pellet assay, while GRASLND overexpression - either via transgene ectopic expression or by endogenous activation via CRISPR-dCas9-VP64 - significantly enhanced cartilage matrix production. GRASLND acts to inhibit IFN-γ by binding to EIF2AK2, and we further demonstrated that GRASLND exhibits a protective effect in engineered cartilage against interferon type II. Our results indicate an important role of GRASLND in regulating stem cell chondrogenesis, as well as its therapeutic potential in the treatment of cartilage-related diseases, such as osteoarthritis.

Genetic Engineering of Mesenchymal Stem Cells for Differential Matrix Deposition on 3D Woven Scaffolds.

Tissue engineering approaches for the repair of osteochondral defects using biomaterial scaffolds and stem cells have remained challenging due to the inherent complexities of inducing cartilage-like matrix and bone-like matrix within the same local environment. Members of the transforming growth factor ß (TGFß) family have been extensively utilized in the engineering of skeletal tissues, but have distinct effects on chondrogenic and osteogenic differentiation of progenitor cells. The goal of this study was to develop a method to direct human bone marrow-derived mesenchymal stem cells (MSCs) to deposit either mineralized matrix or a cartilaginous matrix rich in glycosaminoglycan and type II collagen within the same biochemical environment. This differential induction was performed by culturing cells on engineered three-dimensionally woven poly(ɛ-caprolactone) (PCL) scaffolds in a chondrogenic environment for cartilage-like matrix production while inhibiting TGFß3 signaling through Mothers against DPP homolog 3 (SMAD3) knockdown, in combination with overexpressing RUNX2, to achieve mineralization. The highest levels of mineral deposition and alkaline phosphatase activity were observed on scaffolds with genetically engineered MSCs and exhibited a synergistic effect in response to SMAD3 knockdown and RUNX2 expression. Meanwhile, unmodified MSCs on PCL scaffolds exhibited accumulation of an extracellular matrix rich in glycosaminoglycan and type II collagen in the same biochemical environment. This ability to derive differential matrix deposition in a single culture condition opens new avenues for developing complex tissue replacements for chondral or osteochondral defects.

Regulation of decellularized tissue remodeling via scaffold-mediated lentiviral delivery in anatomically-shaped osteochondral constructs.

Cartilage-derived matrix (CDM) has emerged as a promising scaffold material for tissue engineering of cartilage and bone due to its native chondroinductive capacity and its ability to support endochondral ossification. Because it consists of native tissue, CDM can undergo cellular remodeling, which can promote integration with host tissue and enables it to be degraded and replaced by neotissue over time. However, enzymatic degradation of decellularized tissues can occur unpredictably and may not allow sufficient time for mechanically competent tissue to form, especially in the harsh inflammatory environment of a diseased joint. The goal of the current study was to engineer cartilage and bone constructs with the ability to inhibit aberrant inflammatory processes caused by the cytokine interleukin-1 (IL-1), through scaffold-mediated delivery of lentiviral particles containing a doxycycline-inducible IL-1 receptor antagonist (IL-1Ra) transgene on anatomically-shaped CDM constructs. Additionally, scaffold-mediated lentiviral gene delivery was used to facilitate spatial organization of simultaneous chondrogenic and osteogenic differentiation via site-specific transduction of a single mesenchymal stem cell (MSC) population to overexpress either chondrogenic, transforming growth factor-beta 3 (TGF-ß3), or osteogenic, bone morphogenetic protein-2 (BMP-2), transgenes. Controlled induction of IL-1Ra expression protected CDM hemispheres from inflammation-mediated degradation, and supported robust bone and cartilage tissue formation even in the presence of IL-1. In the absence of inflammatory stimuli, controlled cellular remodeling was exploited as a mechanism for fusing concentric CDM hemispheres overexpressing BMP-2 and TGF-ß3 into a single bi-layered osteochondral construct. Our findings demonstrate that site-specific delivery of inducible and tunable transgenes confers spatial and temporal control over both CDM scaffold remodeling and neotissue composition. Furthermore, these constructs provide a microphysiological in vitro joint organoid model with site-specific, tunable, and inducible protein delivery systems for examining the spatiotemporal response to pro-anabolic and/or inflammatory signaling across the osteochondral interface.